Voltage-gated Ca2+ channels couple membrane depolarization to the influx of Ca2+ into the cell, which initiates a wide variety of biologically important processes including phosphorylation, gene transcription and neurotransmitter release. Because of the importance of these Ca2+ channels for cellular signaling, their activity is subject to numerous forms of regulation. In particular, Cav2.1 (P/Q-type) Ca2+ channels undergo a dual feedback regulation by Ca2+ ions which contributes to short-term plasticity at some synapses. During repetitive stimuli, Cav2.1 Ca2+ currents initially increase (facilitate) and gradually decrease (inactivate). Both facilitation and inactivation are Ca2+-dependent and therefore can be influenced by factors controlling intracellular Ca2+ levels in neurons. Preliminary results presented in this proposal indicate that Ca2+ buffering proteins, which are present at high concentrations in some neurons, and Ca2+ in intracellular stores may be important determinants of Cav2.1 regulation by Ca2+. The experiments outlined in the research plan will test the hypothesis that these factors critically influence the extent to which neuronal Cav2.1 channels are modulated by Ca2+. Whole-cell patch clamp electrophysiology, molecular biology, and Ca2+ imaging techniques will be used to characterize how Ca2+ buffering proteins and intracellular Ca2+ stores affect activity-dependent feedback of Cav2.1 by Ca2+ both in transfected cells and isolated neurons. The findings from this research may reveal novel mechanisms underlying the heterogeneous properties of Cav2.1 channels in neurons and provide new insights into alternative strategies to treat diseases associated with Ca2+ channel defects, such as migraine, epilepsy and ataxia.